In Y eight young mature leaves
In year 1 (Y1), plant roots were washed to remove the soil and the plant was then clamped so that the roots sat in a container of water bubbled with air. The plant was situated on the laboratory bench beside a window with the addition of a small quantity of artificial light during the daylight hours (combined 150–200 μmol/m2/s PAR) from 06:00 to 22:00 h. The following day a section of fine roots was placed in a plastic bag with wet paper towels and the bag was then fixed with tape to a Bremsstrahlung detector aimed at the terminal portion of the fine roots. At 14:00 h an aliquot of 14C sodium bicarbonate solution (4 MBq) was placed into a vial with a screw cap and septa and a saturated oxalic AM630 solution was injected into the vial. The vial was put into a 150 × 150 mm snap lock bag that was placed over the youngest mature leaf at a distally opposite position of the plant to the selected roots (path of approximately 1500 mm). Some Blu-Tack® (Bostik) was placed on the leaf petiole, as close to the leaf blade as possible, and the snap lock closed, with the Blu-Tack® (Bostik) sealing between the snap lock and the petiole. The vial was opened and left for 30 min before removing the bag, releasing any unfixed 14CO2. The intensity of β− particles at the root was assessed by a Bremsstrahlung detector which measured the low-level X-rays produced by the decay at 10-min intervals for 48 h after label application (Black et al., 2012). Lead shielding was used to shield each detector from portions of the plant other than the monitored tissue ensuring that X-rays were counted only from the tissue in question. The following year (Y2) this experiment was repeated in a temperature-controlled room (light levels 150–200 μmol/m2/s PAR from 06:00 to 22:00 h) using two further detectors, one placed at the end of the peduncle at the entry point to the fruit (path of approximately 50 mm) associated with the radiolabelled youngest mature leaf and another at the point rootward where the labelled leaf main cane met the trunk (path of approximately 750 mm). In this instance the radio-label was applied at 09:30 h, to correlate with the timing of other experiments, but otherwise measurements were made as previously described.